Integrins are a family of glycoprotein membrane receptors that mediate cell-matrix and cell-cell interactions. Integrins are heterodimers, consisting of α and β subunits. To date at least 24 distinct integrin heterodimers have been described, including 18α and 8β subunits. Integrins mediate anchorage and migration of cells via specific interaction with different extracellular matrix (ECM) proteins. In addition, cell survival, division and differentiation also rely on effective cell-ECM associations (Morgan et al. 2009, IUBMB Life, 61:731-38).
Integrins α5β1 and αvβ3 are localized in the adhesion contacts of cultured cells. The integrin-ECM complex not only serves to sustain cell-cell and cell-matrix interactions needed for anchorage and migration, the formation of cell-ECM complex also triggers integrin-mediated intra-cellular signaling by recruiting enzymes and adaptors into dynamic complexes inside a cell. The intracellular signals downstream of integrin can influence gene expression, cell survival, differentiation and proliferation. Integrins have been implicated in many pathological conditions such as angiogenesis and tumor progression.
Several integrins were known to interact with fibronectin via the Arg-Gly-Asp (RGD) motif present in fibronectin. These integrins include 5 αv integrins (αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8) and two β1 integrins (α5β1 and α8β1) (Smith, W., 2008, J. Allergy Clin. Immunol. 121:S375-S379). Fibronectin is a high-molecular weight (about 440 kDa) glycoprotein of the extracellular matrix that binds to integrins as well as other extracellular matrix proteins. Fibronectin exists as a dimer of two monomers linked by disulfide bonds at the C-terminus. Each fibronectin monomer has a molecular weight of 230-250 kDa that contains three types of domains: type I, II, and III. Type I and II domains are stabilized by intra-chain disulfide bonds, while type III domains do not contain any disulfide bonds. The absence of disulfide bonds can result in flexibility in the structure of FN type III domain.
Each domain contains a number of modules organized to form functional and protein-binding regions along the length of a fibronectin monomer. For example, modules in type I domains are required for initiation of fibronectin matrix assembly, and modules in both type I and type III domains are important for association with other fibronectin molecules. The RGD motif is located in module 10 of the type III domain (FNIII 10), and constitutes the binding site of fibronectin to integrins α5β1 and αvβ3. The sequence in type III domain module 9 (FNIII 9), especially the PHSRN synergy loop, facilitates the binding of fibronectin to integrin α5β1. Integrin αvβ3 binding of fibronectin does not require the synergistic effect of module 9.
Because integrins' roles in regulating a variety of cellular functions, the feasibility of using integrin antagonists as treatments of diseases has been studied. For example, antagonists for integrin αIIbβ3, the integrin that activates platelet aggregation, were experimented as an anti-coagulant for treating thrombosis-related ischemic vascular diseases (Coller et al., 2008, Blood, 112:3011-25). Disintegrin, such as Rhodostomin, is a family of small protein integrin antagonists naturally found in snake venom that inhibits integrin-mediated platelet aggregation and cell adhesion. Disintegrin, however, is non-specific and highly immunogenic. It competes with fibronectin for binding to β1- and β3-containing integrins on the cell surface and non-discriminatively inhibits the activities of integrins α5β1, αvβ3 and integrin αIIbβ3. Thus, the use of disintegrin as an antagonist for integrins α5β1 and αvβ3 posts a high risk of hemorrhage due to its αIIbβ3 antagonist activity that prevents platelet aggregation and blood clotting.
Similarly, the Yersinia pseudotuberculosis protein invasin is an integrin-binding protein. The bacterial invasin protein facilitates bacteria entry into cells by binding to integrins. The use of invasin as an integrin antagonist is problematic because the bacterial protein is likely highly immunogenic and because the specificity of the invasin protein is not defined.
At high concentrations, FNIII 9-10 to some extent mimics the biological activity of the full-length fibronectin molecule (van der Walle et al., 2002, Protein Engineering 15:1021-24). FNIII9-10 competes with other ECM proteins for binding to integrins. Such truncated fibronectin molecule or fragment comprising the binding domain for integrin, e.g., FNIII 10 or FNIII 9-10, is less immunogenic in human and has higher specificity, as compared with distintegrins, and thus does not cause hemorrhage. However, difficulties exist when using truncated fibronectin as integrin antagonists. One of the difficulties is stability and solubility. Human FNIII 9 or FNIII 9-10 alone is structurally unstable (see van der Walle, supra). Further, FNIII 9-10 at high concentration is insoluble, which presents great challenges to its large scale preparation and production. Therefore, there is a need for a better designed integrin antagonist with low immunogenicity, high specificity, and enhanced stability and solubility.